Viruses are potent cancer-inducing agents. While the clinical effects of cancer are obvious, its cellular and molecular causes are not fully understood. Here we propose to study the cellular entry mechanisms of polyomavirus (Py), a non-enveloped DNA tumor virus that induces cancer in mice. To infect cells, Py binds to a receptor at the plasma membrane and travels to the endoplasmic reticulum (ER) where it hijacks cellular machineries to cross the ER membrane and reach the cytosol. The virus is then transported into the nucleus where transcription and replication of the viral DNA ensue, leading to uncontrolled cell proliferation and cancer. How Py is transported across the ER membrane remains unclear and is a subject of intense interest. Using an in vitro biochemical approach, we propose to identify the ER factor(s) that facilitate the transport of Py from the ER into the cytosol and to clarify the molecular mechanism of this process. Next, we will employ an in vivo cell biological method to probe the physiological role of these ER factors in Py infection. Interactions between pathogens and their respective host cells expound on basic cellular events;thus, Py's engagement of cellular machineries to cross the ER membrane will elucidate fundamental membrane transport processes. Moreover, identifying the cellular targets that Py co-opts during infection may lead to the development of drugs that allow selective interference. As many of Py's structurally-related viruses are human pathogens, such as the JC, BK, and papilloma viruses, the lessons gleaned from the cellular entry mechanism of Py may be applied to a broader spectrum of human diseases.